Role of Muscarinic Acetylcholine Receptors in Adult Neurogenesis and Cholinergic Seizures

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Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors (GPCRs) that mediate important functions in the periphery and in the central nervous systems. In the brain these receptors modulate many processes including learning, locomotion, pain, and reward behaviors. In this work we investigated the role of mAChRs in adult neurogenesis and further clarified the regulation of muscarinic agonist-induced seizures. We first investigated the role of mAChRs in adult neurogenesis in the subventricular zone (SVZ) and the subgranular zone (SGZ). We were unable to detect any modulation of adult neurogenesis by mAChRs. Administration of muscarinic agonists or antagonists did not alter proliferation or viability of adult neural progenitor cells (aNPCs) <italic>in vitro</italic>. Similarly, muscarinic agonists did not alter proliferation or survival of new adult cells <italic>in vivo</italic>. Loss of the predominant mAChR subtype in the forebrain, the M<sub>1</sub> receptor, also caused no alterations in adult neurogenesis <italic>in vitro</italic> or <italic>in vivo</italic>, indicating that the M<sub>1</sub> receptor does not mediate the actions of endogenous acetylcholine on adult neurogenesis. We also investigated the interaction between mAChRs and cannabinoid receptor 1 (CB<sub>1</sub>) in muscarinic agonist pilocarpine-induced seizures. Previous work showed that pilocarpine-induced seizures require activation of M<sub>1</sub> receptors; activation of these receptors also increases release of endocannabinoids which act at CB<sub>1</sub> receptors to reduce excitatory transmission. Using submaximal doses of pilocarpine, we found that pilocarpine-induced seizures were more severe in CB<sub>1</sub> KO mice compared to WT mice. Similarly, pretreatment with CB<sub>1</sub> antagonists also increased pilocarpine seizure severity. In contrast, pretreatment with CB<sub>1</sub> agonists had no effect on pilocarpine seizure severity. These results support the hypothesis that endocannabinoids act at CB<sub>1</sub> receptors to reduce sensitivity to pilocarpine seizures and that endocannabinoid activity following pilocarpine administration is maximal at CB<sub>1</sub> receptors, so exogenous CB<sub>1</sub> receptor agonists cannot further modulate sensitivity to pilocarpine seizures. Because organophosphates require mAChR activity in order to induce seizures, we investigated whether seizures induced by the organophosphate paraoxon also shared the same regulation by M<sub>1</sub> receptors and CB<sub>1</sub> receptors as pilocarpine seizures. We compared seizure behaviors induced by paraoxon in WT and M<sub>1</sub> KO or CB<sub>1</sub> KO mice. We found that, in contrast to pilocarpine seizures, loss of M<sub>1</sub> or CB<sub>1</sub> receptors had no effect on paraoxon seizure severity. CB<sub>1</sub> antagonist or agonist pretreatment also had no effect on paraoxon-induced seizure severity, indicating that CB<sub>1</sub> receptors do not regulate paraoxon seizures. To further explore the difference in M<sub>1</sub> regulation of pilocarpine and paraoxon seizures, we compared the ability of pilocarpine and paraoxon to induce seizure-independent ERK activation in the hippocampus of WT and M<sub>1</sub> KO mice. Seizure-independent activation of ERK by pilocarpine in the hippocampus was dependent on M<sub>1</sub> receptors. However, paraoxon did not activate ERK in the hippocampus when seizure activity was blocked in WT or M<sub>1</sub> KO mice. This suggests that paraoxon administration, unlike pilocarpine, does not strongly activate M<sub>1</sub> receptors in the hippocampus. Altogether these results indicate that, despite a shared requirement for mAChRs to induce seizures, there are significant differences in the regulation of pilocarpine and paraoxon seizures.